The genome of Burkholderia cenocepacia J2315, an epidemic pathogen of cystic fibrosis patients

Bacterial infections of the lungs of cystic fibrosis (CF) patients cause major complications in the treatment of this common genetic disease. Burkholderia cenocepacia infection is particularly problematic since this organism has high levels of antibiotic resistance, making it difficult to eradicate; the resulting chronic infections are associated with severe declines in lung function and increased mortality rates. B. cenocepacia strain J2315 was isolated from a CF patient and is a member of the epidemic ET12 lineage that originated in Canada or the United Kingdom and spread to Europe. The 8.06-Mb genome of this highly transmissible pathogen comprises three circular chromosomes and a plasmid and encodes a broad array of functions typical of this metabolically versatile genus, as well as numerous virulence and drug resistance functions. Although B. cenocepacia strains can be isolated from soil and can be pathogenic to both plants and man, J2315 is representative of a lineage of B. cenocepacia rarely isolated from the environment and which spreads between CF patients. Comparative analysis revealed that ca. 21% of the genome is unique in comparison to other strains of B. cenocepacia, highlighting the genomic plasticity of this species. Pseudogenes in virulence determinants suggest that the pathogenic response of J2315 may have been recently selected to promote persistence in the CF lung. The J2315 genome contains evidence that its unique and highly adapted genetic content has played a significant role in its success as an epidemic CF pathogen.     

Regulation of Hfq mRNA and Protein Levels in Escherichia coli and Pseudomonas aeruginosa by the Burkholderia cenocepacia MtvR sRNA

Small non-coding RNAs (sRNAs) are important players of gene expression regulation in bacterial pathogens. MtvR is a 136-nucleotide long sRNA previously identified in the human pathogen Burkholderia cenocepacia J2315 and with homologues restricted to bacteria of the Burkholderia cepacia complex. In this work we have investigated the effects of expressing MtvR in Escherichia coli and Pseudomonas aeruginosa. Results are presented showing that MtvR negatively regulates the hfq mRNA levels in both bacterial species. In the case of E. coli, this negative regulation is shown to involve binding of MtvR to the 5′-UTR region of the hfq_Ec mRNA. Results presented also show that expression of MtvR in E. coli and P. aeruginosa originates multiple phenotypes, including reduced resistance to selected stresses, biofilm formation ability, and increased susceptibility to various antibiotics.     

Comparative Metabolic Systems Analysis of Pathogenic Burkholderia

Burkholderia cenocepacia and Burkholderia multivorans are opportunistic drug-resistant pathogens that account for the majority of Burkholderia cepacia complex infections in cystic fibrosis patients and also infect other immunocompromised individuals. While they share similar genetic compositions, B. cenocepacia and B. multivorans exhibit important differences in pathogenesis. We have developed reconciled genome-scale metabolic network reconstructions of B. cenocepacia J2315 and B. multivorans ATCC 17616 in parallel (designated iPY1537 and iJB1411, respectively) to compare metabolic abilities and contextualize genetic differences between species. The reconstructions capture the metabolic functions of the two species and give insight into similarities and differences in their virulence and growth capabilities. The two reconstructions have 1,437 reactions in common, and iPY1537 and iJB1411 have 67 and 36 metabolic reactions unique to each, respectively. After curating the extensive reservoir of metabolic genes in Burkholderia, we identified 6 genes essential to growth that are unique to iPY1513 and 13 genes uniquely essential to iJB1411. The reconstructions were refined and validated by comparing in silico growth predictions to in vitro growth capabilities of B. cenocepacia J2315, B. cenocepacia K56-2, and B. multivorans ATCC 17616 on 104 carbon sources. Overall, we identified functional pathways that indicate B. cenocepacia can produce a wider array of virulence factors compared to B. multivorans, which supports the clinical observation that B. cenocepacia is more virulent than B. multivorans. The reconciled reconstructions provide a framework for generating and testing hypotheses on the metabolic and virulence capabilities of these two related emerging pathogens.     

Assessment of three Resistance-Nodulation-Cell Division drug efflux transporters of Burkholderia cenocepacia in intrinsic antibiotic resistance

Background  

Burkholderia cenocepacia are opportunistic Gram-negative bacteria that can cause chronic pulmonary infections in patients with cystic fibrosis. These bacteria demonstrate a high-level of intrinsic antibiotic resistance to most clinically useful antibiotics complicating treatment. We previously identified 14 genes encoding putative Resistance-Nodulation-Cell Division (RND) efflux pumps in the genome of B. cenocepacia J2315, but the contribution of these pumps to the intrinsic drug resistance of this bacterium remains unclear.     

Identification of small RNAs in Mycobacterium smegmatis using heterologous Hfq

Gene regulation by small RNAs (sRNAs) has been extensively studied in various bacteria. However, the presence and roles of sRNAs in mycobacteria remain largely unclear. Immunoprecipitation of RNA chaperone Hfq to enrich for sRNAs is one of the effective methods to isolate sRNAs. However, the lack of an identified mycobacterial hfq restricts the feasibility of this approach. We developed a novel method that takes advantage of the conserved inherent sRNAs-binding capability of heterologous Hfq from Escherichia coli to enrich sRNAs from Mycobacterium smegmatis, a model organism for studying Mycobacterium tuberculosis. We validated 12 trans-encoded and 12 cis-encoded novel sRNAs in M. smegmatis. Many of these sRNAs are differentially expressed at exponential phase compared with stationary phase, suggesting that sRNAs are involved in the growth of mycobacteria. Intriguingly, five of the cis-encoded novel sRNAs target known transposases. Phylogenetic conservation analysis shows that these sRNAs are pathogenicity dependent. We believe that our findings will serve as an important reference for future analysis of sRNAs regulation in mycobacteria and will contribute significantly to the development of sRNAs prediction programs. Moreover, this novel method of using heterologous Hfq for sRNAs enrichment can be of general use for the discovery of bacterial sRNAs in which no endogenous Hfq is identified.     

Improved Electrotransformation and Decreased Antibiotic Resistance of the Cystic Fibrosis Pathogen Burkholderia cenocepacia Strain J2315

The bacterium Burkholderia cenocepacia is pathogenic for sufferers from cystic fibrosis (CF) and certain immunocompromised conditions. The B. cenocepacia strain most frequently isolated from CF patients, and which serves as the reference for CF epidemiology, is J2315. The J2315 genome is split into three chromosomes and one plasmid. The strain was sequenced several years ago, and its annotation has been released recently. This information should allow genetic experimentation with J2315, but two major impediments appear: the poor potential of J2315 to act as a recipient in transformation and conjugation and the high level of resistance it mounts to nearly all antibiotics. Here, we describe modifications to the standard electroporation procedure that allow routine transformation of J2315 by DNA. In addition, we show that deletion of an efflux pump gene and addition of spermine to the medium enhance the sensitivity of J2315 to certain commonly used antibiotics and so allow a wider range of antibiotic resistance genes to be used for selection.Burkholderia cenocepacia is part of the Burkholderia cepacia complex (Bcc), a group of closely related bacteria of soil, water, and roots (41) recently updated to at least 15 related species (42). Bcc displays many interesting features (see reference 27 for a review). Originally discovered as responsible for soft onion rot (3), Bcc species also interact beneficently with plants (see reference 34 for a review) and may degrade pollutants such as phthalate or the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5,-T) (25, 33). But it is the emergence of Bcc as an opportunistic pathogen of people suffering from cystic fibrosis (CF) (19) and immunocompromizing conditions that has drawn most attention to these bacteria. Among Bcc species, Burkholderia multivorans and B. cenocepacia are the most prevalent in the epidemiology of CF. In particular, strains of the ET12 lineage of B. cenocepacia were responsible for a major transcontinental epidemic among CF patients in the 1990s (20), an outbreak aggravated by the high levels of resistance to nearly all antibiotics that characterizes Bcc. Species of the Bcc have large genomes (7 to 9 Mb) composed of two or three chromosomes and one or more plasmids, an unusual genomic organization among bacteria. The first Bcc genome to be sequenced was that of B. cenocepacia J2315 (also known as LMG16656), the type strain of the ET12 lineage and the reference strain for CF epidemiology; the sequence was completed and made available by the Wellcome Trust Sanger Institute in 2003. It revealed three chromosomes of 3.9, 3.2, and 0.9 Mb and a plasmid of 93 kb. The annotation of this genome was released recently (15).The pathogenicity and multipartite genome of B. cenocepacia make it an important subject for both practical and fundamental study. Genetic modification is essential to the success of many such investigations. Unfortunately, J2315 throws up major barriers to genetic manipulation. Standard electrotransformation techniques are ineffective with this strain, as also found elsewhere (26). Conjugal introduction of DNA has proved unreliable despite adaptations (7) that have enabled occasional successes with B. cenocepacia species (9, 40) including J2315 (39) (see also Results below). Besides, the natural resistance of J2315 to antibiotics, high even on the scale of the generally extensive resistance of B. cenocepacia species (31), severely restricts the use of antibiotic resistance in genetic selections. Circumventing these problems by resorting to a proxy strain, B. cenocepacia K56-2, that has not been sequenced and is more permissive to gene transfer (26, 17, 32, 9) runs the risk that results will be of uncertain relevance to J2315.In the context of our general aim to decipher the role of the four replicon-specific ParABS systems of J2315 (6), we have sought to overcome these obstacles. We report here the reproducible electrotransformation of J2315, and we analyze factors that improve its efficiency. We report also our isolation of a J2315 derivative with reduced antibiotic resistance and the broadened selection possibilities this offers. Detailed protocols are provided which should facilitate studies of this pathogen.     

Mutations in Interaction Surfaces Differentially Impact E. coli Hfq Association with Small RNAs and Their mRNA Targets

The RNA chaperone protein Hfq is required for the function of all small RNAs (sRNAs) that regulate mRNA stability or translation by limited base pairing in Escherichia coli. While there have been numerous in vitro studies to characterize Hfq activity and the importance of specific residues, there has been only limited characterization of Hfq mutants in vivo. Here, we use a set of reporters as well as co-immunoprecipitation to examine 14 Hfq mutants expressed from the E. coli chromosome. The majority of the proximal face residues, as expected, were important for the function of sRNAs. The failure of sRNAs to regulate target mRNAs in these mutants can be explained by reduced sRNA accumulation. Two of the proximal mutants, D9A and F39A, acted differently from the others in that they had mixed effects on different sRNA/mRNA pairs and, in the case of F39A, showed differential sRNA accumulation. Mutations of charged residues at the rim of Hfq interfered with positive regulation and gave mixed effects for negative regulation. Some, but not all, sRNAs accumulated to lower levels in rim mutants, suggesting qualitative differences in how individual sRNAs are affected by Hfq. The distal face mutants were expected to disrupt binding of ARN motifs found in mRNAs. They were more defective for positive regulation than negative regulation at low mRNA expression, but the defects could be suppressed by higher levels of mRNA expression. We discuss the implications of these observations for Hfq binding to RNA and mechanisms of action.     

Biochemical and Functional Studies on the Burkholderia cepacia Complex bceN Gene,Encoding a GDP-D-Mannose 4,6-Dehydratase

This work reports the biochemical and functional analysis of the Burkholderia cenocepacia J2315 bceN gene, encoding a protein with GDP-D-mannose 4,6-dehydratase enzyme activity (E.C.4.2.1.47). Data presented indicate that the protein is active when in the tetrameric form, catalyzing the conversion of GDP-D-mannose into GDP-4-keto-6-deoxy-D-mannose. This sugar nucleotide is the intermediary necessary for the biosynthesis of GDP-D-rhamnose, one of the sugar residues of cepacian, the major exopolysaccharide produced by environmental and human, animal and plant pathogenic isolates of the Burkholderia cepacia complex species. V_max and K_m values of 1.5±0.2 µmol.min⁻¹.mg⁻¹ and 1024±123 µM, respectively, were obtained from the kinetic characterization of the B. cenocepacia J2315 BceN protein by NMR spectroscopy, at 25°C and in the presence of 1 mol MgCl₂ per mol of protein. The enzyme activity was strongly inhibited by the substrate, with an estimated K_i of 2913±350 µM. The lack of a functional bceN gene in a mutant derived from B. cepacia IST408 slightly reduced cepacian production. However, in the B. multivorans ATCC17616 with bceN as the single gene in its genome with predicted GMD activity, a bceN mutant did not produce cepacian, indicating that this gene product is required for cepacian biosynthesis.     

Clostridium difficile Hfq can replace Escherichia coli Hfq for most of its function

A gene for the Hfq protein is present in the majority of sequenced bacterial genomes. Its characteristic hexameric ring-like core structure is formed by the highly conserved N-terminal regions. In contrast, the C-terminal forms an extension, which varies in length, lacks homology, and is predicted to be unstructured. In Gram-negative bacteria, Hfq facilitates the pairing of sRNAs with their mRNA target and thus affects gene expression, either positively or negatively, and modulates sRNA degradation. In Gram-positive bacteria, its role is still poorly characterized. Numerous sRNAs have been detected in many Gram-positive bacteria, but it is not yet known whether these sRNAs act in association with Hfq. Compared with all other Hfqs, the C. difficile Hfq exhibits an unusual C-terminal sequence with 75% asparagine and glutamine residues, while the N-terminal core part is more conserved. To gain insight into the functionality of the C. difficile Hfq (Cd-Hfq) protein in processes regulated by sRNAs, we have tested the ability of Cd-Hfq to fulfill the functions of the E. coli Hfq (Ec-Hfq) by examining various functions associated with Hfq in both positive and negative controls of gene expression. We found that Cd-Hfq substitutes for most but not all of the tested functions of the Ec-Hfq protein. We also investigated the role of the C-terminal part of the Hfq proteins. We found that the C-terminal part of both Ec-Hfq and Cd-Hfq is not essential but contributes to some functions of both the E. coli and C. difficile chaperons.